Project Summary Inflammatory bowel disease (IBD), consisting of ulcerative colitis (UC) and Crohn's disease, is a source of substantial morbidity for 1.4 million people in the USA, and it can progress to colon cancer. It is difficult to treat, with costly immunotherapies only inducing remission in less than half of cases. We have been focusing on the role of the semi-essential amino acid, L-arginine (L-Arg) as a complementary and alternative medicine. We have demonstrated mechanisms for beneficial effects of L-Arg in vitro and in colitis. Cationic amino acid transporter 2 (CAT2), the inducible transporter of L-Arg, is upregulated in colonic macrophages in the epithelial injury and inflammation model induced by dextran sulfate sodium (DSS). Oral L-Arg supplementation is effective as a treatment for DSS colitis. In our human studies, tissue CAT2 expression and L-Arg uptake are decreased in UC patients. Consistent with this finding, we show that CAT2-/- mice exhibit defective L-Arg uptake in colon tissues, and an associated worsening of DSS colitis, indicating that we have developed a novel animal model that mimics human UC in terms of disrupted L-Arg availability. In addition to a profound increase in myeloid cell infiltration, CAT2-/- mice also exhibit a shift from IFN-? to IL-17 as the predominant colonic T cell cytokine, a finding that also parallels our results in human UC. CAT2-/- mice have increased colon tumor number and size in the azoxymethane (AOM)-DSS model of colitis-associated carcinoma (CAC). L-Arg is the substrate for arginase or inducible nitric oxide (NO) synthase (iNOS). We have shown that colonic epithelial restitution is dependent on CAT2 and arginase. The latter generates L-ornithine (L-Orn), which can be metabolized by ornithine decarboxylase (ODC) to generate polyamines, or ornithine aminotransferase (OAT) to generate L-proline (L-Pro). Knockdown of OAT, but not ODC, prevented beneficial effects of L-Arg on restitution, implicating OAT in the maintenance of epithelial function. ODC+/- mice exhibit improvement in DSS colitis, with reduced Th1 response, an effect that is dependent on macrophage NO and TGF-?, indicating enhanced regulatory macrophage responses. We hypothesize that benefits of L-Arg in colitis and colitis- associated tumorigenesis depend on CAT2 and downstream effectors to improve epithelial restitution, and regulate innate and adaptive immunity. In our Aims we will determine if: 1) exacerbation of colitis due to deletion of CAT2 is due to a macrophage defect and if this is ameliorated by supplementation of L-Orn or L-Pro in combination with L-Arg; 2.) improvement in colitis in ODC+/- mice is due to a macrophage effect and if it results from enhanced L-Arg availability for iNOS and/or OAT; 3.) accelerated tumorigenesis with CAT2 deletion is due to a macrophage defect and if this process can be beneficially modulated by downregulation of ODC or supplementation of L-Orn or L-Pro in combination with L-Arg. Through investigation of multiple mouse models, epithelial function, and immune responses, these studies seek to provide new macronutrient-based strategies for treatment of IBD and prevention of colitis-associated dysplasia and carcinoma.